Involvement of platelet-activating factor in endotoxin-induced priming of rabbit polymorphonuclear leukocytes
1Department of Physiology, University of Melbourne, Parkville, Vic., Australia.
Abstract:
Endotoxin primes polymorphonuclear leukocytes (PMNs) for increased superoxide anion (O2-) generation in response to the chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (fMLP). We have investigated the role of platelet-activating factor (PAF) in the priming of rabbit PMNs by endotoxin. In non-primed PMNs, fMLP stimulated PAF generation, but none was detected in the supernatant, whereas in primed PMNs PAF generation increased 17-fold and a large proportion of the total PAF formed was released. There was a close relationship between the concentration-response curve for increases in PAF synthesis and that for enhanced O2- generation. The possibility that PAF was causally linked to enhanced O2- generation in primed PMNs was investigated using compounds previously reported to reduce PAF generation, namely the serine protease inhibitor, tosyl-phenylalanine chloromethylketone, and the phospholipase A2 inhibitor, bromophenacylbromide. Both compounds reduced PAF generation and release in endotoxin-primed PMNs and prevented the enhancement of O2- generation. We examined the possibility that PAF acted in an autocrine fashion to enhance O2- generation following its release from fMLP-stimulated PMNs. However, rabbit PMNs, unlike human PMNs, were not primed by exogenous PAF (0.1-1000 nM) over a concentration range extending beyond that covering the concentrations of PAF determined in the supernatants of fMLP-stimulated, endotoxin-primed PMNs. We conclude that PAF release is unlikely to play a role in priming induced by endotoxin in rabbit PMNs, but our data are not inconsistent with a role for cell-associated PAF in this phenomenon. Furthermore, the release of a large amount (80%) of the total PAF from primed, but not non-primed PMNs suggests a cell-cell mediator role for PAF only in primed PMNs.
Insights
Endotoxin primes polymorphonuclear leukocytes (PMNs) for enhanced superoxide anion generation. Platelet-activating factor (PAF) release is increased in primed PMNs, but exogenous PAF does not enhance superoxide generation in rabbits, suggesting cell-associated PAF may play a role.
Area of Science:
- Immunology
- Cellular Biology
- Biochemistry
Background:
- Endotoxin is known to prime polymorphonuclear leukocytes (PMNs) for enhanced superoxide anion (O2-) generation upon stimulation with formyl-methionyl-leucyl-phenylalanine (fMLP).
- Platelet-activating factor (PAF) is a potent lipid mediator involved in inflammatory responses.
- The precise mechanisms by which endotoxin primes PMNs are not fully elucidated.
Purpose of the Study:
- To investigate the role of platelet-activating factor (PAF) in the endotoxin-induced priming of rabbit polymorphonuclear leukocytes (PMNs).
- To determine if PAF generation and release correlate with enhanced superoxide anion (O2-) production in primed PMNs.
- To explore the potential autocrine or cell-cell mediator role of PAF in PMN priming.
Main Methods:
- Rabbit PMNs were primed with endotoxin and stimulated with fMLP.
- PAF generation and release into the supernatant were measured.
- Superoxide anion (O2-) generation was quantified.
- Inhibitors of PAF synthesis (tosyl-phenylalanine chloromethylketone and bromophenacylbromide) were used to assess causality.
- The effect of exogenous PAF on PMN priming was evaluated.
Main Results:
- fMLP stimulated PAF generation in non-primed PMNs, with minimal release into the supernatant.
- Endotoxin-primed PMNs showed a 17-fold increase in PAF generation and significant release of PAF.
- Increased PAF synthesis correlated with enhanced O2- generation in primed PMNs.
- Inhibitors of PAF synthesis reduced both PAF generation/release and O2- generation in primed PMNs.
- Exogenous PAF did not prime rabbit PMNs, even at high concentrations.
Conclusions:
- PAF release is unlikely to be the primary mechanism for endotoxin-induced priming of rabbit PMNs.
- Cell-associated PAF may contribute to the priming phenomenon.
- The significant release of PAF from primed PMNs suggests a potential role as a cell-cell mediator in specific contexts.
